EFFICIENT METHODS FOR THE SYNTHESIS OF [2-15N]GUANOSINE AND 2′-DEOXY[2-15N]GUANOSINE DERIVATIVES

The nucleophilic addition–elimination reaction of 2′,3′,5′-tri-O-acetyl-2-fluoro-O ⁶-[2-(4-nitrophenyl)ethyl]inosine (8) with [¹⁵N]benzylamine in the presence of triethylamine afforded the N ²-benzyl[2-¹⁵N]guanosine derivative (13) in a high yield, which was further converted into the N ²-benzoyl[2-¹⁵N] guanosine derivative by treatment with ruthenium trichloride and tetrabutyl-ammonium periodate. A similar sequence of reactions of 2′,3′,5′-tri-O-acetyl-2-fluoro-O ⁶-[2-(methylthio)ethyl]inosine (9) and the 6-chloro-2-fluoro-9-(β-D-ribofuranosyl)-9H-purine derivative (11), which were respectively prepared from guanosine, with potassium [¹⁵N]phthalimide afforded the N ²-phthaloyl [2-¹⁵N]guanosine derivative (15; 62%) and 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-[¹⁵N]phthalimido-9H-purine (17; 64%), respectively. Compounds 15 and 17 were then efficiently converted into 2′,3′,5′-tri-O-acetyl[2-¹⁵N]guanosine. The corresponding 2′-deoxy derivatives (16 and 18) were also synthesized through similar procedures.     

流感裂解疫苗H3N2-抗H3N2免疫原性复合物滴鼻诱导小鼠黏膜免疫应答的研究

目的研究流感裂解病毒疫苗抗原抗体复合物滴鼻诱生小鼠黏膜免疫应答.方法分别以15μg H3N2、H3N2-CpG、H3N2-鼠抗H3N2及H3N2-PEG滴鼻免疫小鼠,检测肺泡灌洗液抗H3N2 IgA、血清抗H3N2 IgG效价.取免疫小鼠脾细胞,体外抗原刺激,用定量酶联免疫吸附试验(ELISA)检测上清液IFN-γ及IL-4分泌水平.结果H3N2-抗H3N2免疫原性复合物诱生的抗H3N2 IgA效价明显高于H3N2单独免疫组(P＜0.01),而与H3N2-CpG组无显著性差异.此外,复合物诱生的血清抗H3N2也高于H3N2单独免疫组(P＜0.05).H3N2-CpG组诱生的IFN-γ水平明显升高,而其他组之间无明显差异.结论流感病毒血凝素抗原抗体复合物、血凝素抗原加CpG佐剂可以诱生较强的局部黏膜免疫和体液免疫.这两组诱生的IgA效价均明显高于H3N2单独免疫组.另外,H3N2-CpG组小鼠的脾脏细胞经特异性抗原诱导后培养上清液中的IFN-γ水平明显升高.     

Efficient Synthesis of [2-15N]Guanosine and 2′-Deoxy[2′-15N]Guanosine Derivatives Using N-(tert-Butyldimethylsilyl)[15N]Phthalimide as a 15N-Labeling Reagent

Nucleophilic aromatic substitution of 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-fluoro-9 H-purine with N-(tert-butyldimethylsilyl)[ ¹⁵ N]phthalimide in the presence of a catalytic amount of CsF at room temperature in DMF efficiently afforded the 6-chloro-2-[ ¹⁵ N]phthalimidopurine derivative, which was subsequently converted to the [2-¹⁵N]guanosine derivative was also efficiently synthesized through a similar procedure.     

白细胞介素-2和狂犬病毒N蛋白基因重组与表达

研究白细胞介素－２ 与狂犬病毒Ｎ蛋白基因工程产物的免疫活性。采用生物工程技术,结果显示经基因扩增分别获得４００ｂｐ 和１４００ｂｐ 的ＩＬ－２ 和狂犬病毒Ｎ蛋白基因片段,构建了表达载体ＰＬＹ－４ＩＮ,于工程菌中转化后。得到５６ＫＤ重组蛋白产物,该产物具有ＩＬ－２活性,可诱导小鼠抵抗狂犬病毒攻击。     

NMR Studies of N 1- and N 3-(D-2′-Deoxyribofuranosyl) Nucleosides from Ethyl 5-Aminoimidazole-4-Carboxylate

Abstract

The conformation of deoxyribosides derived from N¹ and N³ substitution of the aglycone ethyl 5-aminoimidazole-4-carboxylate (AICE) has been determined by ¹H and ¹³C NMR spectroscopy. The deoxyribose ring shows considerable variation in the form and population of the contributing N and S conformers but the syn/anti ratio is similar to that in the purine analogues.     

枯草芽孢杆菌N2-10的基因组测序和比较基因组分析

【背景】枯草芽孢杆菌N2-10是一株具有较强抑菌能力且能产纤维素酶等多种水解酶的革兰氏阳性菌,在发酵饲料中具有较大的应用潜力。【目的】通过获得枯草芽孢杆菌N2-10的全基因组序列信息,进一步解析菌株次级代谢产物合成基因信息,并通过比较基因组学分析菌株N2-10与模式菌株的差异性,为阐明N2-10抑菌和益生机制提供理论基础。【方法】通过二代Illumina NovaSeq联合三代PacBio Sequel测序平台,对菌株N2-10进行全基因组测序,将测序数据进行基因组组装、基因预测与功能注释,并利用比较基因组学分析N2-10与其他菌株的差异。【结果】菌株N2-10基因组大小为4 036 899 bp,GC含量为43.88%;共编码4 163个编码基因,所有编码基因总长度为3594369bp,编码区总长度占基因组总长度的89.04%;含有85个tRNA、10个5S rRNA、10个16S rRNA、10个23S rRNA,以及2个CRISPR-Cas、1个前噬菌体和6个基因岛;在GO (gene ontolog)、COG (clusters of orthologous groups of...     

Oligonucleotides Incorporating N7-(2′-Deoxy-β-d-erythro-pentofuranosyl)isoguanine

Abstract

The H-phosphonate and the phosphoramidite of N⁷-2′-deoxyisoguanosine (2) were prepared and incorporated into oligonucleotide duplexes. Their base pairing properties were investigated and compared with those of the parent purine nucleosides.     

N4-(3-Methyl-2-Butenyl)-2-(Methylthio)Tubercidin and Its α-Anomer - 7-Deazapurine Nucleosides with Potential Anticytokinin Activity

Abstract

Phase-transfer catalysis of pyrrolo[2,3-d]pyrimidine 4a with the halogenose 5 yields the anomers 6a and 7a. Deprotection with boron trichloride gives the chloro nucleosides 6b and 7b, which are converted into the potential anticytokinin 2 and its α-anomer 3.     

地表UV-B辐射增强对土壤-大豆系统N2O排放的影响

通过大田试验和室外盆栽试验,采用人工增加紫外辐射的方法模拟UV-B辐射增强,用静态箱-气相色谱法测定N_2O排放通量,研究地表UV-B辐射增强对土壤-大豆系统N_2O排放的影响.结果表明:在相同的气象条件和田间管理措施下,UV-B辐射增强没有改变土壤-大豆系统N_2O排放通量的季节性变化规律.但从植株结荚到成熟,UV-B辐射增强降低了土壤-大豆系统N_2O排放通量,进而降低了N_2O的累积排放量.收割实验发现,在分枝开花期,UV-B辐射增强对土壤N_2O排放影响明显,降低了土壤N_2O排放通量;从结荚至鼓粒期,UV-B辐射增强主要通过降低植株地上部分N_2O排放通量来降低土壤-大豆系统的N_2O排放.UV-B辐射增强显著降低了植株的生物量,并影响到植株的氮代谢和土壤NH_4~+-N与微生物氮.UV-B辐射增强可能会导致农田生态系统N_2O排放量降低.     

H9N2禽流感病毒M2 DNA疫苗初免-蛋白加强免疫的保护效果研究

流感病毒M2(基质蛋白2)是A型流感病毒的一个高度保守的蛋白。由于其免疫原性较弱,本研究采用M2 DNA疫苗初免-蛋白加强的策略来考察M2的免疫保护效果。制备A/Chicken/Jiangsu/07/2002(H9N2)流感病毒的M2 DNA疫苗以及经大肠杆菌表达的去除M2跨膜区的M2蛋白即sM2。以SPF级BALB/c小鼠为模型,电击法免疫M2 DNA疫苗,滴鼻法免疫sM2蛋白,免疫间隔三周,并于末次免疫后三周以致死量5LD50流感病毒H9N2攻击小鼠,通过检测小鼠存活率、体重丢失率、肺部病毒滴度及IgG抗体水平等指标来评价免疫的保护效果。实验结果表明,基于M2的疫苗采用DNA疫苗初免蛋白加强免疫二次的免疫程序能诱导较高的特异性抗体,明显减轻小鼠流感病症,提供完全的保护。     

N9N2亚型禽流感病毒ns1基因的融合表达

华南农业大学动物科学学院陈丽、谢青梅等七位研究生和教授对H9N2亚型禽流感病毒NS1基因的研究结果表明:禽流感病毒(AIV)非结构蛋白NS1在病毒粒子中不存在,故可通过检测NS1抗体来鉴别禽流感受病毒感染鸡群和免疫鸡群,将NS1基因和T4等菌体SOC基因在大肠埃希氏菌中融合表达,其融合蛋白可以作为鉴别禽流感免疫鸡群和感染鸡群的检测抗原。     

Translesion Synthesis across 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-γ-HMHP-dA) Adducts by Human and Archebacterial DNA Polymerases

The 1,N⁶-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N⁶-γ-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N⁶-propano group on 1,N⁶-γ-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N⁶-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) β, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N⁶-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol β. Single nucleotide incorporation experiments indicated that although hpol η, κ, and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N⁶-γ-HMHP-dA and detected large amounts of −1 and −2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N⁶-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.     

我国北方几种玉米和设施菜地土壤的N2O和N2排放特征

为了探究旱地土壤施入氮肥后的气态氮(N₂O和N₂)损失规律,本研究通过室内好氧培养试验(60 d,25 ℃,80%孔隙含水量),运用¹⁵N同位素示踪技术,研究了4个玉米地土壤(哈尔滨、沈阳、栾城、寿光)和2个设施菜地土壤(沈阳、寿光)在施入尿素后的氮转化、N₂O和N₂排放动态。试验中尿素添加量为167 mg N·kg^-1,以模拟田间氮肥施用量200 kg N·hm^-2。结果表明: 在4个玉米地土壤中,尿素施用60 d内N₂O累积排放量为寿光(20 mg N·kg^-1)>栾城(14 mg N·kg^-1)>沈阳(5 mg N·kg^-1)>哈尔滨(0.5 mg N·kg^-1),N₂累积排放量为栾城(176 mg N·kg^-1)>沈阳(106 mg N·kg^-1)>寿光(75 mg N·kg^-1)>哈尔滨(12 mg N·kg^-1);在2个设施菜地土壤中,寿光土壤N₂O累积排放量(21 mg N·kg^-1)是沈阳(2 mg N·kg^-1)的10倍,而两个站点N₂累积排放量分别为28和24 mg N·kg^-1。不同土壤N₂O排放占两种气体排放总量的5%～40%,其中寿光土壤(30%～40%)显著高于其他样地土壤(1%～10%)。在土壤排放的N₂O和N₂中,土壤氮库分别贡献了56%和61%,高于添加当季氮肥的贡献率。相关分析表明,N₂O累积排放量与本底土壤pH呈正相关,说明土壤本底pH可能是调控不同旱地土壤N₂O和N₂排放的重要环境因子。在华北碱性土壤区,采用能降低土壤pH值的措施可能具有较好的气态氮减排效果。     

树木N2O排放速率的测定

 采用封闭罩法,对长白山阔叶红松林的几个主要树种——水曲柳 (Fraxinus mandshurica)、红松 (Pinus koraiensis)、毛赤杨（Alnus hirsuta）和椴树（Tilia amurensis）的连体枝叶排放N2O的速率进行了原位测定, 并考察了树木的N2O排放速率与土壤含水量的关系。结果表明：在观测期间,4种树木的N2O排放速率的变化范围分别为：水曲柳9.46～152.85 ng N2O·g－1 DW·h－1、红松1.37～64.51 ng N2O·g－1 DW·h－1、赤     

不同培养条件对粉被虫草产N6-(2-羟乙基)腺苷的影响

N6-(2-羟乙基)腺苷[N6-(2-hydroxyethyl)-Adenosine,HEA],又名茧草菌素,是一种钙离子拮抗剂,具有抗紫外辐射、抗血小板凝结和镇痛等效用,是集医药保健、化妆品等多项开发潜力于一身的生物资源,也是衡量虫草制品质量的一个重要生物活性物质指标。该研究采用高效液相色谱法对菌丝体中HEA进行检测,以HEA的产量为指标,考察不同培养方法、培养基及其组分、前体物和氨基酸对粉被虫草(Cordyceps pruinosaPetch)cp菌株产HEA的影响。结果表明:在沙氏培养基上,静置培养120 d,cp菌株HEA的产量明显优于摇床培养20 d和发酵罐培养3 d的产量,说明随培养时间的延长,cp菌株中HEA的累积会逐步增加。在静置培养条件下,cp菌株在查氏培养基上HEA的产量明显优于沙氏培养基和PD培养基,HEA的产量是沙氏培养基的3.7倍,是PD培养基的4.48倍。以查氏培养基为基础培养基,静置培养120 d进行碳、氮源的筛选中,最有利于cp菌株产HEA的是蔗糖和硝酸钠;在无机盐的筛选中,K2HPO4是促进cp菌株累积HEA最主要的无机盐,其HEA产量较对照(不添加无机盐的查氏培养基)提高了58.2倍;在查氏培养基上添加不同前体物和氨基酸,发现其结构类似物腺苷、次黄嘌呤和腺嘌呤都有促进cp菌株累积HEA的能力,其中腺苷和次黄嘌呤的效果最好,能提高HEA产量60%以上;添加的14种氨基酸中有组氨酸、L-谷氨酸、甘氨酸、天门冬酰胺、丝氨酸、亮氨酸、L-丙氨酸、赖氨酸和精氨酸等9种氨基酸能促进cp菌株产HEA的能力,其中组氨酸是最有利于cp菌株累积HEA的氨基酸,其HEA产量较ck(查氏培养基)提高251.68%,HEA产量达到(45.56±2.8)mg/L,菌丝体中HEA含量达到(4 633.10±65.65)μg/g。     

A Synthesis of the Potent A2 Selective Adenosine Agonist N6-[2-(3,5-Dimethoxyphenyl)-2-(2-Methylphenyl)Ethyl]Adenosine,and Its 5′-N-Ethyl Ribofuranuronamide Derivative

Abstract

A detailed experimental procedure for the synthesis and resolution of 2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethylamine, and its conversion into the titled adenosine reference agonists is given.     

高速逆流色谱分离制备蛹虫草中虫草素和N6-(2-羟乙基)-腺苷

采用高速逆流色谱（HSCCC）技术从蛹虫草子实体粗提物中分离制备高纯度虫草素和N⁶-(2-羟乙基)-腺苷。利用高效液相色谱（HPLC）测定目标产物在溶剂体系中的分配系数,优化HSCCC分离虫草素和N⁶-(2-羟乙基)-腺苷的溶剂体系,确定了以乙酸乙酯-正丁醇-1.5%氨水（1:4:5,V/V/V）为HSCCC的两相溶剂体系,并运用此溶剂体系,上相为固定相,下相为流动相,主机转速850r/min,流动相流速为1.5mL/min,检测波长为254nm条件下进行分离制备,在250min内从200mg蛹虫草子实体粗提物中一步分离得到10.8mg纯度99%的虫草素和6.1mg 纯度98%的N⁶-(2-羟乙基)-腺苷。该方法简便、快速,为虫草素和N⁶-(2-羟乙基)-腺苷的大量制备建立了基础。     

鼎湖山主要森林土壤N2O排放及其对模拟N沉降的响应

研究了鼎湖山生物圈保护区马尾松(Pinus massoniana)林、混交林和季风常绿阔叶林(季风林)土壤N₂O排放特征及其对氮沉降增加的响应。在1999~2002年期间,3种森林土壤N₂O排放速率均表现明显的季节性变化特点,但这种季节性变化因年份和森林类型不同而异,总的来说,3种森林土壤N₂O排放速率呈现夏秋季较高而冬春季较低的变化。土壤N₂O排放速率在3年观测期间的平均值分别为(g·hm^-2·d^-1):14.2±3.1(季风林),5.8±0.9(混交林)和5.1±0.9(马尾松林)。土壤N₂O排放速率与土壤温度之间在季风林呈现显著的指数正相关关系,但在混交林和马尾松林中它们之间的关系则均不明显。经3个月的模拟氮沉降试验后,氮沉降增加对季风林和马尾松林土壤N₂O的排放均具有明显的促进作用,且这种促进作用随氮沉降水平的升高而增强,但对混交林土壤N₂O排放的影响则不明显。     

2N一4N嵌合体小鼠及其在基因功能研究上的应用

2N-4N嵌合体是由二倍体(2N)的胚胎细胞(或ES细胞)与四倍体(4N)的胚胎细胞组构成的一种拯救型嵌合体。这种嵌合体由于4N胚胎细胞独特的胚外组织发育特性,从而可获得完全源自2N细胞成份的仔鼠(或胎儿)。这一特性最终在ES细胞转基因的基因功能研究中极具重要价值。综述了2N-4N嵌合体研究进展及其在基因功能研究领域的应用。